multicrispr: gRNA design for prime editing and parallel targeting of thousands of targets

Targeting the coding genome to introduce single nucleotide deletions/insertions via Crispr/Cas9 technology has become a standard procedure in recent years. It has quickly spawned a multitude of methods such as Prime Editing, Crispr/Cas9 assisted APEX proximity labeling of proteins, or homology directed repair (HDR), for which supporting bioinformatic tools are, however, lagging behind. New applications often require specific guide-RNA (gRNA) design functionality, and a generic gRNA design tool is critically missing. Here we review gRNA designer software and introduce multicrispr, an R based tool intended to design individual gRNAs as well as gRNA libraries targeting many genomic loci in parallel. The package is easy to use, detects, scores and filters gRNAs on both efficiency and specificity, visualizes and aggregates results per target or Crispr/Cas9 sequence, and finally returns both genomic ranges as well as sequences of preferred, off target-free gRNAs. In order to be generic, multicrispr defines and implements a genomic arithmetics framework as a basis for facile adaptation to techniques yet to arise. Its performance and new gRNA design concepts such as target set specific filtering for gRNA libraries render multicrispr the tool of choice when dealing with screening-like approaches.